The in vitro system using cell culture has been widely used as a primary screening tool for evaluating anti-HIV activity of microbicides. However, there is clearly a need to develop a vaginal and cervical tissue-based in vitro system to test the cytotoxicity and antiviral activity in the context of the complete tissue matrix. We have recently developed a cervical and vaginal tissue-derived organ culture which mimics in vivo situation and has been used as a model to test antiviral compounds, such as PMA, for its ability to block HIV transmission across the mucosa of cervical tissue. The overall objective of the proposed project is to validate the antiviral properties and cytotoxicity of innovative combinations of UC781, a non-nucleoside reverse transcriptase (RT) inhibitor, and other compounds with diverse mechanisms of action virucidal activity in cervical and vaginal tissue-based organ culture. Specific aims of the project are: (1) To determine the cytotoxicity of combination microbicides with the broad anti-HIV activity on the vaginal and cervical tissues in an organ culture model. The compounds with the highest anti-HIV activity in the presence of excipients from Project 1 and Project 3 will be tested for cytotoxicity in the organ culture model. Histology and immunostaining of immune and non-immune cellular markers in tissues will be used to examine the cytotoxic effect of the microbicides; (2) To evaluate the effect of microbicides on transmission of HIV across the cervical tissue using an organ culture model. The combination microbicides in excipient with the highest anti-HIV activity from Projects 1 and 3, and lowest cytotoxicity (Specific Aim 1) will be tested for its ability to block HIV transmission across the mucosa by measuring the infectivity of the transmitted virus in the CMAGI assay an measuring viral RNA in the tissue by in situ hybridization; and (3) To examine the penetration of microbicides across the vaginal and cervical epithelium. The tissue will be examined for the migration of radiolabeled UC781 and other microbicides across the tissue to determine the mechanism of action.